The outstanding therapeutic success of As2O3 in a subtype of acute myelocytic leukemia (AML), the acute promyelocytic leukemia (APL), prompted widespread effort to extend this therapy to other malignancies. We and others determined that, through generation of reactive oxygen species (ROS), As2O3 induced apoptosis of APL cells. We also showed that degradation of PML-RARa was not a crucial event in the pro-apoptotic program of As2O3, but high ROS production ability was. We thus postulate that by enhancing the ROS-generating capacity through combination therapies we will be able to effectively target non-APL AML for apoptosis. Membrane localized NADPH oxidase and mitochondria are the main sources of ROS production. AML cells share lineage derivation with polymorphonuclear cells, which use NADPH oxidase generated ROS to kill bacteria. We showed that As2O3 treatment of AML cells increases the expression, without causing activation, of a crucial component of NADPH oxidase, p47phox. Our preliminary results show that a combination of therapeutically achievable concentration of As2O3 with fenretinide (4-HPR), a retinoid approved for clinical trials, synergizes in causing apoptosis in AML cells which express detectable (basal or As2O3-induced) NADPH oxidase components and extends survival of AML bearing SCID/NOD mice. Under physiological conditions ROS generated as a byproduct of mitochondrial respiration are detoxified by glutathione-enzymes, glutathione peroxidase (GPx), glutathione-s-transferase ? (GST?) and glutathione reductase (GR). Inhibition of the activities of these enzymes combined with increased ROS generation due to malfunction of the mitochondrial respiration in cancer cells, should generate levels of ROS that are beyond tolerable, causing mitochondria-mediated apoptosis of AML cells. Indeed, a combination of As2O3, which inhibits GPx, with ethacrynic acid (a clinically used diuretic drug) and its new derivative, which we have developed, and which inhibit GST? and GR, synergistically induce apoptosis in AML cells. This effect is independent of NADPH oxidase expression. We propose to study the mechanisms underlying these findings and their effect in primary AML and in vivo (animal models) in 4 specific aims. In aim 1 we will study the mechanism of As2O3 induced NADPH oxidase expression and its activation by 4-HPR through a ceramide dependent pathway. In aim 2 we will study the mechanism of the synergy of apoptosis induction by As2O3 and ethacrynic acid, and its derivative, through inhibition of ROS degradation and decrease of an antiapoptotic protein Mcl-1. In aim 3 we will study the apoptotic effects of As2O3 and 4-HPR or As2O3 and ethacrynic acid in primary AML samples. Importantly, we will investigate the possibility of using NADPH oxidase/myeloperoxidase or GST?/catalase levels in primary human AML cells as potential predictive markers of patient stratification to sensitive or resistant to As2O3/4-HPR or As2O3/ethacrynic acid therapies, respectively. In specific aim 4 we will test the efficacy of the combination treatments in xenografts of human AML. If further studies confirm that these treatment combinations are indeed effective in inducing apoptosis in AML cell lines and primary cells, and that they have therapeutic effect in xenografts, without excessive toxicity, because of the current clinical use of each of the tested compounds, it will be possible to rapidly translate these finding into clinical trials. PUBLIC HEALTH RELEVANCE: Arsenic trioxide (As2O3) is a drug used world-wide that induces complete clinical remission in 90% of APL patients without significant toxicity. Our therapeutic design includes the use of 4 HPR, a drug already in clinical trial, that activates a membrane system for the production of ROS and the use of new non-diuretic derivatives of ethyacrynic acid which block enzymes that diminish ROS and markedly enhanceAs2O3 induced leukemic cell death by several mechanisms. Our goal will be to utilize As2O3 in combination with 4 HPR or EA analogs in clinical trials in patients with refractory, secondary or in elderly patients with acute myelogenous leukemia.